Exp Ⅴ 1. Enzyme-linked Immunosorbent Assay （ ELISA) 2. Complement fixation test (CFT)

1. Exp Ⅴ 1. Enzyme-linked Immunosorbent Assay（ELISA)2. Complement fixation test (CFT)

2. 1. Enzyme-linked Immunosorbent Assay （ELISA)

3. Immunological labeling techniques • Definition: Ag-Ab reactions are combined with labeling techniques. Known Ag or Ab is labeled with radioisotope, enzyme or fluorescein and unknown Ab or Ag are indirectly detected by labeled molecules. • Classification: • Radioimmunoassay(RIA): 131I，32P • Immunofluorescence technique: FITC，PE • Enzyme Immunoassay: HRP，AP

4. Immunological labeling techniques 3. Labeling techniques： Direct labeling techniques: Each Ag or Ab Indirect labeling techniques: Secondary Ab

5. Isotype of antibody: Immunize Rabbit Mouse IgG1 Rabbit anti-mouse IgG1 Secondary Ab

6. Immunofluorescence technique：

7. Enzyme ImmunoAssay (EIA) • Ag-Ab reactions with enzyme-labeled Ag or Ab. • horseradish peroxidase(HRP)，alkaline phosphatase (AP) • Characteristics: • High specificity: Ag-Ab reaction • High sensitivity: enzyme catalytic reaction (pg level) • Qulitative or quantitative assay: Color change or OD value • Classification: • ELISA: Soluble Ag or Ab • Immunochemistry: Ag in tissues or in the surface of cells

8. Immunohistochemistry

9. Enzyme-Linked ImmunoSorbent Assay (ELISA) 1.Definition: Unknown Ab or Ag in blood or culture medium are detected by enzyme-labeled Ag or Ab. • Indirect ELISA: • Known Ag, enzyme-labeled secondary Ab • Unknown Ab • Sandwich ELISA: • Known Ab, enzyme-labeled Ab • Unknown, soluble Ag • Competitive ELISA: • Known Ab or Ag, enzyme-labeled Ab or Ag • Unknown Ag or Ab 2.Classification:

10. Enzyme-Linked ImmunoSorbent Assay (ELISA) 3. Principles (Indirect ELISA for example):

12. Experiment:Assay of hemolysin by Indirect ELISA——Qualitative assay

13. Materials: • Defribinated SRBC —— Ag • Rabbit anti-SRBC Ab(hemolysin) —— Primary Ab • HRP-labeled goat anti-rabbit IgG —— Secondary Ab • Coating buffer：0.05M pH9.6 bicarbonate buffer • Washing buffer：0.01M PBS(pH7.4) containing 0.05% Tween 20 • Substrate buffer： pH5.0 phosphate-citrate buffer solution • Substrate solution：OPD 10mg,Substrate buffer 25ml,30% H2O2 40μL • Microwell plate

14. Methods Defibrated SRBC Add 3mL N.S and mix 2000rpm，5min 1. Preparation of Ag: Add 3mL N.S and mix 2000rpm，5min Take 2mL packed SRBC Add 2mL DDW and shatter RBC Dilute with coating buffer in a ratio of 1：400 Add 100μL of Ag to each well of ELISA plate 2. Coating Ag: 4℃，in a humidified box, 18h

15. Discard the Ag solution in ELISA plate(4wells/group) Methods Wash the plate 3 times(3min each time) 3. Add test serum 1 2 3 4 Sign and add 100μL of solution to each well Blank Negative Positive Test 37℃ for 45min in a humidified box Discard the solution in ELISA plate 4. Add secondary Ab Wash the plate 3 times(3min each time) Add 100μL of HRP-labeled secondary Ab to each well 37℃ for 30min in a humidified box Discard the solution in ELISA plate 5. Showing color Wash the plate 3 times(3min each time) Add 100μL of substrate solution to each well Show color in dark for10min 6. Observe the result

16. Anticipated results • Positive：yellow • Negative：blank 1 2 3 4 Neg Neg Pos Pos

17. Attentions • Wash thoroughly and avoid cross-contamination • Add samples in turn and no bubbles in the bottom of the wells • Coating and incubation should be performed in humidified box

18. 2. Complement fixation Test(CFT)

19. Complement fixation reaction: Ag-Ab reaction in the presence of complement and with SRBC and hemolysin (anti-SRBC Ab) as an indicator system. Definition

20. Principle Two systems, five components in CFT: • Test system: known Ag or Ab, unknown Ab or Ag and quantitative complement. • Indicator system: SRBC and hemolysin

21. 1 2 Ag+Unknown serum Complement SRBC+hemolysin 1 2 1 2 Step 1 Formation of Ag-Ab complex Step 2 Complement fixation and exhaustion Step 3 Lysis of SRBC

22. Patient’s serum Y Y Y Y Y Y Y Y Ab Positive No Ab Negative Ag Ag Ag Ag Y

23. Material • Ag: Lytic products from typhiod • Test serum: 56℃，30min • Complement: from guinea pig • 2% SRBC • Hemolysin

24. Serum control Ag control Complement control SRBC control Test Methods 1 2 3 4 5 … … … … … … 0.2mL 0.2mL Test serum Ag … … … … … … 0.2mL 0.2mL Complement … … 0.2mL 0.2mL 0.2mL 0.2mL N.S … … 0.2mL 0.4mL 0.8mL 0.2mL Shake，incubate in water bath at 37℃ for 15min … … Hemolysin 0.2mL 0.2mL 0.2mL 0.2mL 0.2mL 0.2mL SRBC 0.2mL 0.2mL 0.2mL Shake，incubate in water bath at 37℃ for 15min

25. Anticipated results 1．Hemolysis: clear, red No hemolysis: turbid, ruddy Hemolysis No hemolysis 2.

26. Attentions • Shake SRBC before use • Pipettes for different reagents should not be confused